Acute and subchronic toxicity as well as mutagenic evaluation of essential oil from turmeric (Curcuma longa L)

Food and Chemical Toxicology 53 (2013) 52–61

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Acute and subchronic toxicity as well as mutagenic evaluation of essential oil from turmeric (Curcuma longa L) Vijayasteltar B Liju, Kottarapat Jeena, Ramadasan Kuttan ⇑ Department of Biochemistry, Amala Cancer Research Centre, Amala Nagar 680555, Thrissur, Kerala, India

a r t i c l e

i n f o

Article history: Received 26 July 2012 Accepted 18 November 2012 Available online 29 November 2012 Keywords: Turmeric essential oil Subchronic toxicity Wistar rats Mutagenecity Genotoxicity

a b s t r a c t The present study investigated the acute, subchronic and genotoxicity of turmeric essential oil (TEO) from Curcuma longa L. Acute administration of TEO was done as single dose up to 5 g of TEO per kg body weight and subchronic toxicity study for thirteen weeks was done by daily oral administration of TEO at doses 0.1, 0.25 and 0.5 g/kg b.wt. in Wistar rats. There were no mortality, adverse clinical signs or changes in body weight; water and food consumption during acute as well as subchronic toxicity studies. Indicators of hepatic function such as aspartate aminotransferase (AST), alanine amino transferase (ALT) and alkaline phosphatase (ALP) were unchanged in treated animals compared to untreated animals. Oral administration of TEO for 13 weeks did not alter total cholesterol, triglycerides, markers of renal function, serum electrolyte parameters and histopathology of tissues. TEO did not produce any mutagenicity to Salmonella typhimurium TA-98, TA-100, TA-102 and TA-1535 with or without metabolic activation. Administration of TEO to rats (1 g/kg b.wt.) for 14 days did not produce any chromosome aberration or micronuclei in rat bone marrow cells and did not produce any DNA damage as seen by comet assay confirming the non toxicity of TEO. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Spices containing essential oils are important for their medicinal properties. They impart a pleasant aroma and taste, there by forming major components of many food items, soft drinks and beverages. The rhizome of Curcuma longa L. belonging to the family Zingiberaceae is extensively and traditionally used in many Asian countries to enhance the food quality, color, flavor and antioxidant properties (Krishnaswamy, 2008; Ruby et al., 1995). Different cultures have also used this spice to treat a myriad of diseases and ailments. Turmeric essential oil (TEO) is prepared from the rhizome of turmeric by steam distillation (Liju et al., 2011). TEO is different from oleoresin of turmeric where curcuminoids are the major compounds while ar-turmerone is the major constituent of TEO (Sacchetti et al., 2005). Several medicinal and pharmacological properties such as antifungal, insect repellent, anti-bacterial, anti-platelet and anti-mutagenic activities of TEO have been reported (Negi et al., 1999; Jayaprakasha et al., 2002; Sacchetti et al., 2005; Prakash et al., 2011). TEO also possess anti-inflammatory, antioxidant, anti-arthritic and antinociceptive properties ⇑ Corresponding author. Tel./fax: +91 487 230 7968. E-mail addresses: [email protected], amalacancerresearch@gmail. com (R. Kuttan). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.11.027

(Jayaprakasha et al., 2002; Funk et al., 2010; Liju et al., 2011). It has shown efficacy in neuroprotective activity against cerebral ischemia and attenuation of delayed neuronal death via a caspase- dependent pathaway (Jain et al., 2007; Dohare et al., 2008; Rathore et al., 2008). The chemopreventive efficacy of TEO has been reported against submucous fibrosis in humans (Deepa Das et al., 2010). It also acts against benzo[a]pyrene induced DNA damage in vitro in oral mucosa cells (Hastak et al., 1997). TEO can improve the bioavailability of curcumin after oral administration in humans (Antony et al., 2008). Food and Drug Administration (FDA) approved TEO usage as food additive and is listed as Generally Recognized As Safe (GRAS), while the FDAs GRAS list does not include the dosage of TEO. TEO is usually used in aroma therapy and the recommended dose is 1–2 drops/day. Anti-arthritic evaluation of TEO in mice was carried out at doses of 560 mg/kg b.wt. (Funk et al., 2010), but no systematic studies on the toxicity of TEO has been reported. In the present study we have evaluated acute, subchronic and genotoxic effects of TEO on rats. We have looked into several parameters including body weight changes, food and water consumption, hematological parameters, organ toxicity and histopathology of various tissues. Moreover in vitro bacterial reverse mutation test (Ames test) was done to evaluate the mutagenicity of TEO and the genotoxic effect of TEO was studied by micronuclei, chromosomal aberration and comet assay.

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was collected and weighed. Similarly volume of water consumption for 24 h was calculated for each group of animals.

2. Materials and methods 2.1. Turmeric essential oil

2.6. Urinalysis TEO isolated by steam distillation from the rhizome of turmeric was supplied by Kancore Ingredients Limited, Angamali, Kerala, India. The color and appearance of TEO was pale yellow liquid and stored at 4 °C away from direct light (Sample No. TONE-00321). TEO was found to be stable at these conditions through out the experiment. An initial study in our laboratory on the composition of TEO by GC– MS indicated that the main components are ar-turmerone (61.79%) and curlone (12.48%). Other major and minor ingredients in this essential oil are ar-curcumene (6.11%), phenol (3.45%), zingiberene (2.98%), a-sesquiphellandrene (2.81%), 1-ethyl4-isobutylbenzene (2.62%), a–bisabolene (1.48%), benzene (1.48%), benzaldehyde (1.44%), 1,2,3,5-tetramethyl-benzene (1.42%), silane (0.84%), and 4-methyl-carbanilonitrile (1.09%) (Liju et al., 2011). 2.2. Animals All animal experiments were conducted after getting prior permission from Institutional Animal Ethics Committee and which followed the instructions prescribed by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India. Young adult male (Average weight 200 g) and female (Average weight 140 g) Wistar rats 7–8 weeks old were purchased from Small Animal Breeding Station, Kerala Agricultural University, Mannuthy, Kerala, India. They were housed in the animal house of Amala Cancer Research Centre, in well ventilated sterile polypropylene cages. Each cage contained five rats of the same sex. They were maintained at a controlled temperature of 22 ± 2 °C and relative humidity 60 ± 10% and provided 12 h light/dark cycles. Experiments were started after acclimatizing the rats for one week. They were fed with normal pelleted rat chow (Sai Durga Feeds and Foods, Bangalore, India) and water ad libitum. The composition of the feed is- maize 36%, rice bran 34%, rice polish 4.97%, cotton seed extract 4%, soybean meal 10%, groundnut extract 4%, dried yeast 2%, salt 5%, mineral mixture 0.025%, vitamin 0.01%, mean energy (kcal/kg) 3600, pellet size 12 mm.

Five male and five female Wistar rats were administered single dose of 5 g/kg b.wt. TEO by oral gavage and were transferred to metabolic cages (one rats per cage). After 24 h, the urine samples were collected, during which the rats were deprived of food to avoid solid debris pollution, but were allowed water ad libitum. The collected urine was measured and analyzed for glucose and albumin with Magistik-GP urinalysis strip. Tests were done to determine pH and presence of ketone bodies, keto acids and microscopy of the urine sediment (optical microscope) was also done. Results were compared with untreated animals. 2.7. Ophthalmic observations Five male and five female Wistar rats were administered with single dose of 5 g/ kg b.wt. TEO for the ophthalmic study. After 24 h, ophthalmic examination of the anterior portion of the eye following dilation of the pupil with a mydriatic agent was done using an ophthalmoscope in the presence of a veterinary doctor. The cornea and retinal area were observed and compared with normal animals. 2.8. Subchronic toxicity study Fifty healthy rats were divided into 5 groups as given below. Dosage selection was based on the therapeutic dosage which was 10 mg/kg/day in human studies (Joshi et al., 2003) and 100 mg/kg for animal studies (Chandra and Gupta, 1972). Group

Treatment

Number and sex

I II III IV V

Untreated Vehicle control (paraffin oil) Turmeric essential oil (0.1 g/kg.b.wt.) Turmeric essential oil (0.25 g/kg.b.wt.) Turmeric essential oil (0.5 g/kg.b.wt.)

5 5 5 5 5

males males males males males

and and and and and

5 5 5 5 5

females females females females females

2.3. Chemicals Nutrient broth was purchased from Hi-media laboratories, Mumbai. Agar agar, biotin, NADP, glucose-6-phosphate, dimethyl sulphoxide and sodium azide (NaN3) were obtained from Sisco Research Laboratories, Mumbai, India. N-methyl-N0 -nitro-N-nitrosoguanidine (MNNG), 4-nitro-O-phenylenediamine (NPD), 2-acetamidofluorene (2-AAF), high melting point agarose, low melting point agarose and propidium iodide were purchased from Sigma Chemicals (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) (Case no. 67–68-5) liquid paraffin (CAS No. 8012–95-1), May Grunwald and Geimsa stain were purchased from Merck Specialties Private Ltd., Mumbai, India. Phenobarbitone (Gardenal R 60, Batch No.B03007) was purchased from Nicholas-Piramal India Ltd., Gujarat, India. Nicotinamide adenine dinucleotide phosphate reduced (NADPH) was obtained from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. Tobacco was purchased from local market. Magistik-GP urinalysis strips were purchased from Peerless Biotech Pvt. Ltd., Chennai, India. All other chemicals were of analytical grade. L-histidine,

TEO was administered once daily in the morning by oral gavage and continued for 13 weeks. The animals were monitored for clinical and behavioral symptoms such as diarrhea, immobility, neuromuscular problems and mortality during the first day and thereafter for 13 weeks. After 13 weeks, the animals were sacrificed under light ether anesthesia. Blood was collected by direct heart puncture. Necropsies of the animals were performed in the presence of a veterinary doctor, and observations were recorded. 2.9. Food and water consumption The quantity of food and water consumed was recorded for each group of animals every five days during the course of the experiment. 2.10. Body and organ weight

2.4. Preparation of turmeric essential oil for oral feeding TEO dissolved in paraffin oil was administered to the rats at different doses in 1 ml. It was prepared fresh every time. Paraffin oil was chosen because of its inert nature and easy solubility of TEO. The control animals were given 1 ml of paraffin oil every day.

The weight of each rat was recorded on the first day and at 5 days intervals throughout the course of the study and mean body weights were calculated. The weight of the brain, liver, stomach, kidney and spleen were recorded and expressed in relation to the final body weight. 2.11. Hematology and clinical chemistry analysis

2.5. Acute toxicity studies on turmeric essential oil Initial studies were done to check whether TEO produced any mortality and adverse reactions when given at maximum concentration recommended. Forty Wistar rats were divided into the following groups. For grouping, animals with similar weight and sex were grouped together. Group

Treatment

Number and sex

I II III IV

Untreated Turmeric essential oil (1 g/kg.b.wt.) Turmeric essential oil (2.5 g/kg.b.wt.) Turmeric essential oil (5 g/kg.b.wt.)

5 5 5 5

males males males males

and and and and

5 5 5 5

females females females females

TEO was administered as single dose through oral gavage and rats were monitored for 14 days. The body weight, mortality, clinical, behavioral symptoms and ophthalmic observations were noted. The food consumption was determined by placing known quantity of the feed in the cage and after 24 h, remaining feed

Blood was collected in EDTA tubes and analyzed for hematological parameters. Total white blood cells were measured after diluting the blood in Turk’s fluid and counting them using a hemocytometer (Nelson and Morrie, 1984). For differential leukocyte count, blood smear was prepared on a clean glass slide, stained with Leishman’s stain and various types of cells were counted manually using a microscope (Pal and Parvati, 2003). Platelet count was determined by diluting the blood with Rees Ecker diluting fluid and counted using a hemocytometer (Pal and Parvati, 2003). Total RBC count was measured by diluting the blood with Dacie’s fluid and counted using a counting chamber (Pal and Parvati, 2003). Hemoglobin content (Hb) was measured by cyanmethaemoglobin (Drabkin’s) method using kit purchased from Agappe Diagnostics, Thane, India. A part of the blood was collected in nonheparinized tubes and serum was separated after centrifugation at 5000 rpm for 10 min which was used for the following investigations. Total bilirubin was determined by Jendrassik-Diazotized sulphanilic acid method (Jendrassik and Gróf, 1938). Alkaline phosphatase (ALP) was estimated by p-nitrophenyl phosphate (PNPP) hydrolysis (Mc Comb and Bowers, 1979) and alanine amino transferase (ALT) as well as aspartate aminotransferase (AST) by kinetic method using kits supplied by Raichem using Merck Microlab300 auto analyzer. Albumin was determined based on its reaction with bromocresol green and

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total protein concentration was determined by biuret method (Nelson and Morrie, 1984). Markers of kidney function such as creatinine and blood urea nitrogen were estimated by Jaffe-Kinetic and urease method respectively (Pearlman and Lee, 1974). Total cholesterol was estimated by CHOD–PAP (cholesterol oxidase–phenol + aminophenazone) enzymatic method (Jaffe, 2003; Deeg and Ziegenhorn, 1983), triglyceride was estimated by GPO–PAP (glycerol-3-phosphate oxidase–phenol + aminophenazone) method (Cole et al., 1997) and HDL cholesterol was determined after precipitation with phosphotungistic. VLDL cholesterol was estimated by the Friedewald equation (VLDL = triglyceride/5) and LDL cholesterol by calculation (LDL = total cholesterol–(HDL + VLDL)). Serum sodium, potassium and bicarbonate were estimated using Flame photometer 129 ion selective electrolyte analyzer. Chloride was estimated by mercurous thiocyanate method using a kit from Raichem. 2.12. Histopathological analysis A portion of the selected organs (liver, kidney, spleen, brain, stomach and intestine) of normal, control and treated group (500 mg/kg of TEO) were fixed in 10% neutral buffered formalin. Embedded organs tissue samples were cut into slices of 2–4 lm and stained with hematoxylin-eosin and the sections were observed under light microscope (40). 2.13. Mutagenicity studies Evaluation of the ability to induce reverse mutation at the histidine loci in four Salmonella typhimurium strains of TA 98, TA 100, TA 102 and TA 1535 (Ames test) was conducted according to standard procedures (Ames et al., 1975). Due to unavailability of TA 1537, which is uvr mutant, it was not used in this study and has been replaced by TA 102. Mutagenicity of TEO was done by plate incorporation method in the presence and absence of an exogenous metabolic activation system. Mutagenicity of TEO was assayed at three doses (0.1, 1, 3 mg/plate) in triplicates. Sodium azide/plate (5 lg) dissolved in distilled sterile water was used as positive control for strain TA 98, TA 100 and TA 102. For strain TA 1535, 20 lg nitro-o- phenylendiamine/plate dissolved in distilled water was used as positive control. In the case of S9 mix activated group, acetamidofluorene (20 lg) was used as positive control. A plate without drug and mutagens was used as negative control and 20 ll DMSO was used as vehicle control. Two ml of top agar layer (0.6% agar and 0.5% NaCl) containing S. typhimurium strains, 0.5 mM histidine/biotin solution and different concentrations of TEO were shaken well and poured onto the surface of 25 ml of minimal agar. After the overlay solidified, the plates (triplicate) were inverted and incubated for 48 h at 37 °C and revertant colonies counted using colony counter. Rat liver microsomal enzyme was used for metabolic activation of mutagen in vitro (Jayaprakasha et al., 2002). Microsome P450 enzymes was induced in rat liver by oral administration of 0.1% phenobarbital dissolved in water for 4 days. The animals were sacrificed on the 5th day. Livers were excised aseptically and microsomal S9 fraction was prepared by centrifuging the homogenate at 9000g for 15 min. Activation mixture was prepared by mixing S9 mix (500 ll) with sodium phosphate buffer (0.2 M, pH 7.4), NADP (0.1 M), glucose 6 phosphate (1 M, pH 7.4), MgCl2–KCl (10 ll) in presence of mutagen, 2-acetamidoflourene (20 lg/plate) or different concentrations of TEO and bacterial strains TA 98 and TA 100 and incubated at 37 °C for 45 min. Further, it was mixed with 2 ml of molten top agar supplemented with histidine and biotin (0.05 mM). The mixture was shaken well and poured onto the surface of 25 ml of minimal agar. After the overlay solidified, the plates (triplicate) were inverted and incubated for 48 h at 37 °C. The mutagenic response was evaluated by counting manually the revertant colonies per plate and compared with the control groups. The test substance is considered to be mutagenic if there is a three fold increase in the tester strains when compared to the negative controls. 2.14. Effect of TEO on micronuclei formation Twelve animals were divided into 4 groups having 3 animals in each group. Group I: untreated male, group II: untreated female, group III: treated male, group IV: treated female, group III and group IV were treated with 1 g/kg b.wt. TEO in paraffin oil by oral gavage for 2 weeks. One hour after last drug administration animals were sacrificed using ether anesthesia. Bone marrow cell from each femur were flushed into phosphate buffered saline (PBS) containing 10% FCS. Tubes were centrifuged at 3000 rpm for 10 min and the cell pellet was collected and smeared onto a clean glass slide. Four slides were prepared from each animal and stained with May Grunwald Geimsa (MGG) and observed under oil immersion objective. Two thousand polychromatic (PCE) and corresponding normochromatic erythrocytes (NCE) were scored for the presence of micronuclei from each animal (Schmid, 1975). 2.15. Effect TEO on chromosomal aberrations Six male and six female Wistar rats were divided into 4 groups having 3 animals in each group. Group I: untreated male, group II: untreated female, group III: treated male and group IV: treated female. TEO (1 g/kg b.wt.) was administered by oral

gavage to group III and IV for 14 days. 1 h after last TEO administration animals were sacrificed. All the treated animals were injected intra peritoneally with colchicine (2 mg/kg b.wt, in saline) 90 min before sacrifice to cause mitotic arrest. Bone marrow cell from each femur were flushed into phosphate buffered saline containing 10% FCS. Cell button was separated by centrifugation and 5 ml of 0.075 M KCL (37 °C) was added to each tube and incubated for 30 min. Tubes were centrifuged again and 5 ml (3 ml drop wise and 2 ml at a stretch) of ice cold methanol-acetic acid mixture (3:1) was added and centrifuged again and cell pellet was separated. The cell pellet was dropped into a chilled clean glass slide (4 drops/slide) from a height of 60 cm and slides were air dried and kept in the dark for 5 days for maturation. Four slides were prepared from each animal and stained with Geimsa and observed under oil immersion and screened for metaphase chromosomes. A minimum of 100 metaphase spread were scored for aberrations (Savage, 1983).

2.16. Analysis of genotoxic effect of TEO by comet assay Six male and six female Wistar rats were divided into 4 groups having 3 animals in each group. Group I: untreated male, group II: untreated female, group III: treated male and group IV: treated female. 1 mg/kg b.wt. TEO was administered by oral gavage to group III and group IV for 2 weeks. On the 14 day after 1 h after TEO administration animals were sacrificed. The DNA damages in the bone marrow, spleen and small intestine cells were measured as single strand-breaks using alkaline single cell gel electrophoresis (comet assay) (Singh, 2000). The cells were flushed into Hank’s balanced salt solution (HBSS) and diluted as 50000–100000 cells/ml. The mixture of 10 ll of cells and 200 ll of 0.8% low melting agarose was layered onto precoated slides, containing 1% normal melting point agarose and then covered with a cover slip. The slides were placed in the chilled lysing solution (2.5 M NaCl, 0.1 M EDTA, 0.01 M Tris HCl- pH 10, 1% DMSO and 1% Triton X 100) for 1.5 h at 4 °C followed by alkaline buffer (pH > 13) for 20 min. Electrophoresis was carried out for 20 min, at 25 V and 300 mA. The slides were stained with 50 ll of propidium iodide (20 lg/ml) and visualized using fluorescence microscope. Images were captured and a minimum of 50 comets per slide, in triplicates for a group were analyzed using the software ‘CASP’ which gives Olive tail moment (OTM) directly.

2.17. Statistical analysis The values are expressed as mean ± SD. The statistical significance was compared between control and experimental groups by one way analysis of variance (ANOVA) followed by appropriate post hoc test (Dunnet multiple comparison test) using Graph pad in Stat software (version 3.05). Data of TEO treated animals were compared with untreated animals. Vehicle control groups were compared with untreated groups.

3. Results 3.1. Acute toxicity studies on turmeric essential oil The administration of TEO at different doses such as 1–5 g/ kg.b.wt did not produce any mortality. The body weight (Fig. 1), clinical signs and food and water consumption (Fig. 2) did not differ when compared with normal group during the period of study. 3.2. Urinalysis There were no significant changes in urine volume and pH (6.5–7.5) in the treated animals when compared with untreated animals. Microscopy of the urinary sediment did not reveal any calcium or phosphate crystals and relevant changes. Urinary glucose, albumin, ketobodies and ketoacids were absent in both treated and untreated animals. 3.3. Ophthalmic observations Ophthalmoscopic observations did not reveal any treatment related changes suggestive of corneal ulcer or retinal vascularity. There was no compression of retinal vessels nor extra branching of vessels could be observed in treated groups and hence common lesions like glaucoma and intraocular inflammatory changes were ruled out.

V.B Liju et al. / Food and Chemical Toxicology 53 (2013) 52–61

Fig. 1. Effect of acute administration of turmeric essential oil (5 g/kg b.wt.) on body weight of male and female rats.

Fig. 2. Effect of acute administration of turmeric essential oil (5 g/kg b.wt.) on food consumption pattern of male and female rats.

Fig. 3a. Effect of subchronic administration of turmeric essential oil on body weight of male rats.

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3.4. Subchronic toxicity study 3.4.1. General conditions and behavior General conditions and behavior of rats were not adversely affected by the TEO administration at doses of 0.1, 0.25 and 0.5 g/ kg b.wt. for 13 weeks. There was no mortality in any of the groups. The animals were healthy and no signs of toxicity were observed during the period of study.

3.4.2. Body weight Administration of TEO for 13 weeks did not produce any abnormal change in the body weight of male and female rats when compared to normal rats (Figs. 3a and 3b). 3.4.3. Food and water consumption Administration of TEO did not produce any difference in the food consumption of male and female rats when compared to nor-

Fig. 3b. Effect of subchronic administration of turmeric essential oil on body weight of female rats.

Fig. 4. Effect of subchronic administration of turmeric essential oil on food consumption pattern of male and female rats.

Table 1 Effect of subchronic (13 weeks) administration of turmeric essential oil on relative organ weight. Group

Liver

Spleen

Lungs

Kidney

Brain

Stomach

Male Untreated Controla 100 mg/kg 250 mg/kg 500 mg/kg

3.89 ± 0.61 4.05 ± 0.38 4.06 ± 0.29 4.07 ± 0.29 4.26 ± 0.19

0.32 ± 0.02 0.29 ± 0.06 0.33 ± 0.05 0.28 ± 0.11 0.30 ±.0.03

0.53 ± 0.18 0.59 ± 0.17 0.51 ± 0.05 0.69 ± 0.24 0.62 ± 0.23

0.72 ± 0.07 0.77 ± 0.06 0.74 ± 0.05 0.81 ± 0.08 0.78 ± 0.08

0.67 ± 0.02 0.61 ± 0.05 0.58 ± 0.03 0.64 ± 0.06 0.59 ± 0.07

0.55 ± 0.02 0.56 ± 0.07 0.61 ± 0.06 0.58 ± 0.05 0.57 ± 0.05

Female Untreated Controla 100 mg/kg 250 mg/kg 500 mg/kg

4.29 ± 0.31 4.54 ± 0.24 4.81 ± 0.56 4.49 ± 0.41 4.58 ± 0.68

0.31 ± 0.03 0.27 ± 0.07 0.29 ± 0.05 0.32 ± 0.07 0.26 ± 0.03

0.77 ± 0.19 0.79 ± 0.11 0.88 ± 0.10 0.81 ± 0.10 0.82 ± 0.09

0.87 ± 0.13 0.89 ± 0.06 0.96 ± 0.04 0.85 ± 0.08 0.99 ± 0.16

0.78 ± 0.01 0.86 ± 0.21 0.83 ± 0.09 0.90 ± 0.12 1.03 ± 0.08

0.73 ± 0.09 0.75 ± 0.22 0.77 ± 0.07 0.70 ± 0.10 0.79 ± 0.08

Values are mean ± standard deviation of 5 animals/sex and expressed as the organ weight/g of body weight. a Vehicle (paraffin oil).

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V.B Liju et al. / Food and Chemical Toxicology 53 (2013) 52–61 Table 2 Effect of subchronic administration of turmeric essential oil on hematological parameters. Group Male Untreated Controla 100 mg/kg 250 mg/kg 500 mg/kg Female Untreated Controla 100 mg/kg 250 mg/kg 500 mg/kg

Hb (g/dl)

RBC (106)

Platelet (106)

WBC (mm3)

14.91 ± 1.52 15.44 ± 1.05 15.84 ± 1.59 14.99 ± 1.68 14.69 ± 1.91

9.11 ± 0.66 8.84 ± 0.32 8.26 ± 0.48 8.12 ± 0.76 8.00 ± 0.79

644.72 ± 33.16 675.50 ± 41.32 611.20 ± 42.85 628.40 ± 44.35 656.40 ± 45.91

9956 ± 245.96 9566 ± 236.14 9620 ± 305.37 9870 ± 454.97 9854 ± 374.01

13.84 ± 0.29 13.67 ± 0.48 13.45 ± 1.01 13.37 ± 1.71 14.41 ± 0.70

6.78 ± 0.61 6.87 ± 0.61 6.41 ± 0.32 6.60 ± 0.44 6.50 ± 0.36

526.60 ± 25.80 572.20 ± 41.10 577.80 ± 41.37 550.20 ± 49.76 578.20 ± 54.84

7658 ± 291.20 7479 ± 255.60 7050 ± 447.21 7073 ± 471.32 7510 ± 230.22

Values are mean ± standard deviation of 5 animals/sex. a Vehicle (paraffin oil).

Table 3 Effect of subchronic administration of turmeric essential oil on liver function test. Group

Total bilirubin (mg/dl)

ALT (U/L)

AST (U/L)

ALP (U/L)

Total protein (g/dl)

Male Untreated Controla 100 mg/kg 250 mg/kg 500 mg/kg

0.44 ± 0.22 0.46 ± 0.29 0.59 ± 0.28 0.47 ± 0.12 0.53 ± 0.08

68.4 ± 06.5 73.8 ± 12.5 76.8 ± 11.3 71.0 ± 10.8 77.8 ± 12.2

182.0 ± 15.1 188.6 ± 13.5 163.8 ± 35.0 162.8 ± 33.4 165.2 ± 41.1

336.2 ± 26.8 345.3 ± 21.2 316.4 ± 09.3 303.8 ± 28.0 292.6 ± 20.0

8.0 ± 0.5 6.9 ± 0.5 6.9 ± 0.3 6.9 ± 0.6 6.8 ± 0.2

Female Untreated Controla 100 mg/kg 250 mg/kg 500 mg/kg

0.39 ± 0.19 0.43 ± 0.05 0.41 ± 0.09 0.48 ± 0.12 0.60 ± 0.11

086.4 ± 10.5 075.8 ± 09.4 072.0 ± 08.2 092.2 ± 0 5.0 066.8 ± 0 2.8

195.4 ± 11.0 182.0 ± 41.6 159.4 ± 37.7 166.2 ± 28.3 153.5 ± 34.4

284.0 ± 40.6 297.7 ± 34.8 300.6 ± 03.7 242.0 ± 14.2 345.0 ± 27.4

7.0 ± 0.3 7.6 ± 0.3 6.9 ± 0.2 7.1 ± 0.4 6.7 ± 0.3

ALT., alanine aminotransferase; AST., aspartate aminotransferase; ALP., alkaline phosphatase. Values are mean ± standard deviation of 5 animals/sex. a Vehicle (paraffin oil).

Table 4 Effect of subchronic administration of turmeric essential oil on renal function test. Group

Blood urea nitrogen (mg/dl)

Serum creatinine (mg/dl)

Male Untreated Controla 100 mg/kg 250 mg/kg 500 mg/kg

55.0 ± 2.9 50.6 ± 4.7 61.0 ± 1.9 64.2 ± 3.4 63.4 ± 3.1

0.68 ± 0.08 0.62 ± 0.04 0.56 ± 0.05 0.59 ± 0.06 0.56 ± 0.03

Female Untreated Controla 100 mg/kg 250 mg/kg 500 mg/kg

77.0 ± 5.9 75.1 ± 5.3 66.6 ± 4.7 60.6 ± 1.1 67.5 ± 1.3

0.62 ± 0.04 0.68 ± 0.04 0.59 ± 0.04 0.60 ± 0.08 0.59 ± 0.06

Values are mean ± standard deviation of 5 animals/sex. a Vehicle (paraffin oil).

mal animals (Fig. 4). The average food intake of male rats was nearly 15 g/day/animal and female was 10 g/day/animal. Similarly, the water consumption did not alter in treated groups when compared to normal animals. 3.4.4. Necropsy Observations of necropsy of TEO treated animals were found to be normal, lacking in any apparent pathological abnormalities in any of the treated groups.

Table 5 Effect of subchronic administration of turmeric essential oil on serum electrolytes. Group

Bicarbonate (mEq/L)

Sodium (mEq/L)

Potassium (mEq/L)

Chloride (mEq/L)

Male Untreated Controla 100 mg/kg 250 mg/kg 500 mg/kg

25.8 ± 0.8 21.8 ± 3.1 20.8 ± 1.6 21.8 ± 1.3 19.0 ± 1.6

147.8 ± 1.66 163.2 ± 10.4 176.8 ± 13.0 157.0 ± 13.6 156.4 ± 16.6

5.5 ± 0.4 7.5 ± 0.4 5.7 ± 1.0 5.9 ± 0.9 5.5 ± 0.9

105.2 ± 0.47 108.8 ± 16.2 114.0 ± 12.3 102.0 ± 05.9 115.8 ± 03.2

Female Untreated Controla 100 mg/kg 250 mg/kg 500 mg/kg

21.6 ± 3.2 21.4 ± 3.2 22.0 ± 2.1 21.2 ± 2.4 21.5 ± 3.9

139.9 ± 1.68 136.8 ± 21.9 167.2 ± 21.8 152.0 ± 10.6 171.5 ± 11.6

4.9 ± 1.1 7.4 ± 0.5 6.5 ± 1.0 5.9 ± 0.4 4.9 ± 0.9

101.3 ± 01.8 111.2 ± 07.2 106.8 ± 08.8 104.4 ± 11.5 101.5 ± 03.5

Values are mean ± standard deviation of 5 animals/sex. a Vehicle (paraffin oil).

3.4.5. Organ weight No significant changes were observed in organ weight of liver, kidney, spleen and stomach with respect to body mass in TEO fed animals when compared with untreated animals (Table 1).

3.4.6. Hematological parameters The hematological parameters such as hemoglobin, RBC count, platelet count, total and differential leukocytes count were

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unaltered in TEO treated animals compared to untreated animals (Table 2). 3.4.7. Serum biochemical parameters TEO did not produce any significant changes in serum enzymes. Hepatic function parameters such as ALT, AST, ALP, total bilirubin, total protein and A/G ratio content were not altered in TEO treated animals of both sexes (Table 3). The renal function parameters such as blood urea and serum creatinine did not show any variation in treated animals compared to untreated group (Table 4). There was no significant change in the levels of serum electrolytes such as chloride, potassium, sodium and bicarbonate (Table 5) after TEO treatment. There was also no change in cholesterol, LDL and VLDL cholesterol levels. All the treated male and female groups showed a slight decrease in the levels of triglycerides (Table 6). 3.4.8. Histopathological analysis Histopathological examination of the organs such as liver, kidney, brain, spleen, stomach and intestine of animals treated with TEO did not show any differences when compared with untreated groups indicating that TEO treatment up to a concentration of 0.5 g/kg b.wt. for 13 weeks did not show any adverse toxicological effects in these organs (Fig. 5). 3.5. Mutagenicity of turmeric essential oil TEO up to a concentration of 3000 lg/plate in the absence and presence of S9 mix did not produce any revertants when compared with negative control in S. typhimurium strains (Table 7). The positive control containing known mutagens induced increase in mean number of revertant colonies in each strain, while vehicle control did not show any increase in revertant colonies. 3.6. Effect of TEO on micronuclei formation No significant micronuclei formations were observed in both male and female in TEO treated bone marrow cells. The percentages of micronucleated PCE and NCE of male and female in untreated groups were 0.61 ± 0.12 and 0.74 ± 0.07 and that of treated group male and female were 0.82 ± 0.09 and 0.94 ± 0.11 which was not significant. 3.7. Effect of TEO on chromosomal aberrations Chromosomal aberrations in untreated male and female rats were 1.41 ± 0.67 and 1.67 ± 0.24. In the TEO treated (1 g/kg b.wt.)

male and female rats aberrations were 1.82 ± 0.43 and 2.0 ± 0.66 after 14 days which indicates that TEO administration did not produce any significant chromosomal aberration. 3.8. Analysis of genotoxic effect of TEO by single cell gel (comet) assay After 14 days administration of TEO (1 g/kg b.wt.) there was no significant olive tail moment in the bone marrow, spleen and intestine of the treated male and female Wistar rats (Fig. 6) when compared with untreated groups indicating TEO did not produce any DNA damage. 4. Discussion TEO requires deeper evaluation for their efficacy and safety due to its growing demand on reported medicinal uses. A computerized toxicity prediction reported that many of the TEO components are non toxic (Balaji and Chempakam, 2010). Currently, we conducted an acute toxicity, 13 weeks subchronic toxicity and genotoxicity study for evaluating the safety of TEO at different doses. The major ingredient of TEO determined by GC/MS is reported to be arurmerone and its bioavailability was reported to be 13% (Liju et al., 2011; Jayaprakasha et al., 2002). Literature survey indicated that the toxicity of TEO which is different from oleoresin in its composition has not been systematically evaluated. The acute oral toxicity study of TEO was done at 5 g/kg in rats and up to this concentration there were no changes in body weight, behavior, ophthalmic, urinalysis and food and water consumption when compared with untreated group. Changes in body weight have been used as an indicator of adverse effects of drugs and chemicals (Tofovic and Jackson, 1999). Since there were no significant changes in the general behavior, body weight, and food and water intake of rats in the treated groups as compared to the untreated group after TEO administration for 13 weeks; it could be concluded that oral administration of TEO had no effect on the growth and functions of rats at the concentration studied. The haematopoietic system is one of the most sensitive parameters to assess the toxicity of drugs in humans and animals (Rahman et al., 2001). Present study indicated that there was no significant difference in hemoglobin, RBC count, platelet count, total and differential leukocytes count between the untreated and TEO treated groups indicating that TEO had no effects on the circulating blood cells or on their production. Any damage to the liver results in elevations of both ALT and AST in the blood. In addition, ALT found in the serum is taken as a first sign of cell and liver damage (Rahman et al., 2001; Hilaly et al., 2004). Creatinine is known as a good indicator of renal

Table 6 Effect of subchronic administration of turmeric essential oil on lipid profile. Group

Cholesterol (mg/dl)

Triglycerides (mg/dl)

HDL (mg/dl)

LDL (mg/dl)

VLDL (mg/dl)

Male Untreated Controla 100 mg/kg 250 mg/kg 500 mg/kg

77.4 ± 7.2 82.2 ± 6.9 70.2 ± 5.7 68.0 ± 2.4 71.6 ± 4.6

137.5 ± 21.0 129.4 ± 21.1 97.6 ± 05.0 99.4 ± 07.3 98.4 ± 04.8

32.8 ± 3.4 38.0 ± 3.6 33.6 ± 5.3 30.8 ± 1.3 33.8 ± 3.4

18.6 ± 3.4 18.6 ± 2.3 19.4 ± 2.8 17.8 ± 1.8 18.6 ± 1.7

26.0 ± 4.9 25.6 ± 4.2 19.2 ± 0.8 19.4 ± 1.1 19.2 ± 0.8

Female Untreated Controla 100 mg/kg 250 mg/kg 500 mg/kg

75.6 ± 9.4 87.0 ± 4.0 67.4 ± 2.7 69.2 ± 3.5 69.0 ± 1.8

114.2 ± 31.9 141.6 ± 10.5 96.8 ± 5.2 112.6 ± 9.5 95.8 ± 5.9

32.2 ± 3.4 42.6 ± 3.5 30.2 ± 2.4 30.0 ± 1.6 33.3 ± 2.1

20.6 ± 6.8 16.6 ± 1.7 18.2 ± 1.1 17.6 ± 1.9 17.8 ± 1.3

22.8 ± 6.3 27.8 ± 2.2 19.0 ± 1.2 22.0 ± 1.9 19.0 ± 0.8

Values are mean ± standard deviation of 5 animals/sex. a Vehicle (paraffin oil).

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Fig. 5. Histopathological analysis of organs treated with turmeric essential oil (0.5 g/kg body weight). (A) liver; (B) kidney; (C) brain; (D) spleen; (E) stomach; (F) intestine.

function i.e., rises in creatinine means there is obvious damage to functional nephrons (Rahman et al., 2001; Lameire et al., 2005). There was no significant difference in ALP, ALT, AST, creatinine, blood urea, total bilirubin, total protein and A/G ratio in TEO treated animals when compared to control. These results suggest that

TEO did not alter the renal and hepatic function. The present study also revealed that serum electrolytes did not alter after treatment with TEO. Liver is the site of cholesterol degradation, glucose synthesis and generates free glucose into the blood from hepatic glycogen

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Table 7 Mutagenic study of turmeric essential oil in the S. typhimurium reverse using mutation assay. S. typhimurium strains Doses of turmeric essential oil

TA 98

TA 100

TA102

TA1535

Without S9-mix

3000 lg/plate 1000 lg/plate 100 lg/plate Negative controla DMSO (Vehicle control) Positive controlb

29.4 ± 7.2 36.8 ± 8.4 43.2 ± 6.1 40.9 ± 8.6 35.3 ± 7.9 733 ± 34.6

92.1 ± 16.3 107.4 ± 12.8 97.5 ± 17.3 102.3 ± 21.3 95.7 ± 23.2 542.7 ± 51.3

117.3 ± 22.1 123.1 ± 13.0 110.4 ± 14.7 112.2 ± 13.4 119.3 ± 12.3 494.4 ± 62.6

27.2 ± 5.3 25.3 ± 4.1 30.3 ± 7.4 26.5 ± 8.2 31.8 ± 8.8 711.2 ± 52.3

With S9-mix

3000 lg/plate 1000 lg/plate 100 lg/plate Negative controla DMSO (vehicle control) Positive controlb

43.6 ± 6.6 51.3 ± 8.3 55.4 ± 8.2 47.7 ± 8.4 52.9 ± 9.1 698 ± 52.8

137.2 ± 29.1 131.3 ± 22.2 144.6 ± 34.4 151.8 ± 20.1 143.3 ± 17.3 728.4 ± 66.5

139 ± 12.5 127.4 ± 24.3 134.6 ± 18.7 140.1 ± 23.2 133.3 ± 14.4 580.7 ± 39.3

37.3 ± 2.3 33.8 ± 2.4 40.4 ± 2.9 38.5 ± 3.1 42.1 ± 2.2 576.2 ± 42.9

The values are mean ± standard deviation of 3 different determinations. a Without mutagen and drug. b Sodium azide was used as mutagen in TA 98, TA 100 and TA 102 and nitro-phenylendiamine was used in TA 1535. Acetamidofluorene was used as mutagen in studies involving S9 activities.

Fig. 6. Comet assay for the assessment of genotoxicity of turmeric essential oil in bone marrow, intestine and spleen cells of (male) Wistar rats. (A) bone marrow; (B) intestine; (C) spleen.

stores (Kaplan et al., 1995). In the present study, lowering of triglycerides was observed which was not significant. No changes were observed in cholesterol, LDL, and VLDL levels suggestive that the TEO had no effects on the lipid and carbohydrate metabolism of the rats. The microscopic evaluation of the organs of treated rats group supported the safety of the TEO. Due to the presence of terpenoids present in TEO it is considered to have significant antioxidant activity (Liju et al., 2011) and

has been reported to have chemopreventive activity (Deepa Das et al., 2010). Moreover TEO has been reported to inhibit oral submucosa fibrosis in humans (Hastak et al., 1997). It also has been reported to have significant anti-inflammatory, antinociceptive activity and anti-arthritic activity (Liju et al., 2011; Funk et al., 2010). A combination of several of the fractions that contain the TEO was more effective than the curcuminoids at inhibiting PGE2 (Lantz et al., 2005). It has been reported that TEO enhances the

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bioavailability of curcumin in human (BCM-95) as well as clinical study is ongoing to assess the efficacy and safety of BCM-95 in oral premalignant lesions as well as cervical cancers (Antony et al., 2008; Anand et al., 2008). These results indicate that TEO has a significant medicinal value which has not been exploited yet. TEO (0.1–3 mg/plate) did not produce any revertants during Ames test, indicating that there was no significant dose related mutagenicity of the TEO either with or without metabolic activation. Genotoxic substances are potentially known to be mutagenic or carcinogenic. Exposure of cells to genotoxic substances damage chromosomes of the mitotic spindle leading to the formation of micronuclei. Genotoxic studies of TEO such as micronuclei formation, chromosomal aberrations and genomic DNA damage analysis by comet assay have revealed that there was no genotoxic effect after 2 weeks oral administration of 1 g/kg b.wt. TEO. 5. Conclusions Present study reports the non toxicity of TEO given orally up to a concentration of 0.5 g/kg b.wt. for 13 weeks. TEO did not produce any toxicity as seen from biochemical studies, body weight changes, consumption of food and water as well as histopathology. TEO did not produce any mutagenicity as seen from the S. typhimurium reversion assay. TEO administration did not produce any genotoxicity to rats as seen from the micronuclei, chromosomal aberration in the bone marrow cells as well as from the single cell gel electrophoresis (comet assay). The results of the present study indicate that the TEO is safe in rats (NOAEL) up to an oral dose of 0.5 g/kg b.wt. for 13 weeks. Conflict of Interest The author (s) declared no conflicts of interest with respect to the authorship and/or publication of the article. Acknowledgement Authors are thankful to Spice Board of India, for providing financial assistance to carry out this work. References Ames, B.N., Mccann, J., Yamasaki, E., 1975. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res. 31 (6), 347–364. Anand, P., Thomas, S.G., Kunnumakkara, K.B., Sundaram, C., Harikumar, K.B., Sung, B., Tharakan, S.T., Misra, K., Priyadarsini, I.K., Rajasekharan, K.N., Aggarwal, B.B., 2008. Biological activities of curcumin and its analogues (Congeners) made by man and mother nature. Biochem. Pharmacol. 76 (11), 1590–1611. Antony, B., Merina, B., Iyer, V.S., Judy, N., Lennertz, K., Joyal, S., 2008. A pilot crossover study to evaluate human oral bioavailability of BCM-95 CG (Biocurcumax), a novel bioenhanced preparation of curcumin. Indian J. Pharm. Sci. 70 (4), 445– 449. Balaji, S., Chempakam, B., 2010. Toxicity prediction of compounds from turmeric (Cucuma longa L). Food Chem. Toxicol. 48 (10), 2951–2959. Chandra, D., Gupta, S.S., 1972. Anti-inflammatory and anti-arthritic activity of volatile oil of Curcuma longa (Haldi). Indian J. Med. Res. 60 (1), 138–142. Cole, T.G., Klotzsch, S.G., McNamara, J., 1997. Measurement of triglyceride concentration. In: Riafi, N., Warnick, G.R., Dominiczak, M.H. (Eds.), Handbook of Lipoprotein Testing. AACC Press, Washington, pp. 115–126. Deeg, R., Ziegenhorn, J., 1983. Kinetic enzymatic method for automated determination of total cholesterol in serum. Clin. Chem. 29, 1798–1802.

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